Afrose, Afrina, White, Edward, Howes, Tony,George, Graeme ...€¦ · 5 HPMC 0-0.8%, Pl F127 0-1.8%, leucine 0-1.5% and mannitol 0-9%. They were magnetically stirred until complete

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Afrose, Afrina, White, Edward, Howes, Tony, George, Graeme, Rashid,Abdur, Rintoul, Llew, & Islam, Nazrul(2018)Preparation of ibuprofen microparticles by antisolvent precipitation crystal-lization technique: Characterization, formulation, and in vitro performance.Journal of Pharmaceutical Sciences, 107 (12), pp. 3060-3069.

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https://doi.org/10.1016/j.xphs.2018.07.030

1

Preparation of ibuprofen microparticles by anti-solvent precipitation

crystallization (APC) technique: characterization, formulation and in-vitro

performance

Afrina Afrose1,2

, Edward T White3, Tony Howes

3, Graeme George

2,4, Abdur Rashid

5, Llew

Rintoul4, and Nazrul Islam

1,2,*

1Pharmacy Discipline, School of Clinical Sciences, Faculty of Health, Queensland University of

Technology, Brisbane, QLD

2Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane,

QLD

3School of Chemical Engineering, The University of Queensland, Brisbane, QLD.

4School of Chemistry, Physics and Mechanical Engineering, Science and Engineering Faculty,

Queensland University of Technology, Brisbane, QLD

5Md Abdur Rashid, Assistant Professor, Department of Pharmaceutics, School of Pharmacy,

King Khalid University, Guraiger, Abha-62529, Kingdom of Saudi Arabia.

* The corresponding author

Dr. Nazrul Islam

Pharmacy Discipline, School of Clinical Sciences, Faculty of Health, Queensland University of

Technology, Brisbane, QLD.

E-mail: [email protected]

Phone No. +61 07 31381899

Page 1 of 33 Journal of Pharmaceutical Sciences

2

Abstract

This study demonstrates the preparation and characterization of ibuprofen (IBP)

microparticles with some excipients by a controlled crystallization technique with improved

dissolution performance. Using the optimum concentrations Pluronic F127 (Pl F127),

hydroxypropyl methyl cellulose (HPMC), D-mannitol and L-leucine in aqueous ethanol, the

IBP microparticles were prepared. The dissolution tests were performed in phosphate buffer

saline (PBS) using a USP dissolution tester at 37°C. The Raman spectroscopy was used to

investigate the interactions and distribution of the IBP with the additives in the microcrystals.

The prepared IBP microparticles showed higher dissolution compared to that of the smaller sized

original IBP particles. The Raman data revealed that the excipients with a large number of

hydroxyl groups distributed around the IBP particle in the crystal, enhanced the dissolution of

the drug by increasing the drug-solvent interaction presumably through hydrogen bonding. The

Raman mapping technique gave an insight into the enhanced dissolution behaviour of the

prepared IBP microparticles and such information will be useful for developing pharmaceutical

formulations of hydrophobic drugs. The controlled crystallization was a useful technique to

prepare complex crystals of IBP microparticles along with other additives to achieve the enhanced

dissolution profile.

Key words: Ibuprofen, crystallinity, Raman mapping, excipients, dissolution.

Page 2 of 33Journal of Pharmaceutical Sciences

3

1. Introduction

Antisolvent precipitation crystallization (APC) of particles is a technique that provides the

framework for an appropriate design of micro/nanoparticles to produce engineered particles with

desired physicochemical properties to meet the requirements in their practical applications1.

Addition of additives such as mannitol (morphology modifier, discrete particle former), leucine

(dispersibility enhancer), pluronic (crystal size inhibitor) and hypromellose (HPMC, as

crystal/agglomerate growth inhibitor) in crystallizing drug particles have been studied 2-7

. Using

Ibuprofen (IBP) as a model drug, this study attempted to prepare IBP micro/nanoparticles with

these additives for their characterization and to understand the impact of these excipients in the

formation and dissolution performances of the active drug.

Ibuprofen, a non-steroidal anti-inflammatory drug, is used as various dosage forms in the

treatment of various diseases. This drug is poorly water soluble and thereby the rate of

dissolution from the currently available dosage forms are limited that leads to poor

bioavailability at high dose. Despite the fact that the IBP is a commonly used high dose

therapeutic medicine, its poor solubility in aqueous solutions lessens the dissolution and

absorption rates 8. Crystals of IBU prepared by conventional methods and subsequent

micronization by milling are very difficult to reduce in particle size for improving the solubility.

Dry milling is a commonly used technique to reduce particle size and most of the time particles

tend to deform rather than fragment during the milling process. The heat generated during dry

milling cause partial melting (as this drug has a low melting point) of the milled particles and

thereby produce a large number of amorphous particles that tend to form large agglomerates 9.

The conventional micronization process (i.e. milling, homogenization) with high energy input

incorporates undesirable particle shape, size, surface charge modifications, and decreased

crystallinity that affect the physicochemical properties.

Raman microscopy, a non-destructive analytical technique is used in pharmaceutical

product design for visualization of component distribution in dosage forms10,11

such as tablets12

,

solid dispersion13

and powder mixtures14

. Our study demonstrates an anti-solvent crystallization


4

process for the preparation of IBP microparticles with some excipients (HPMC, mannitol,

leucine and pluronic) to understand their distribution in the microcrystals and subsequent

dissolution behaviour of the active drug. In this study, we utilized Raman Micro-Spectroscopy

for mapping of the individual excipients in a powder mixture to identify the distribution of active

drug among other additives, and their effects on the dissolution of the IBP from the prepared

microcrystals.

2. Material and methods

2.1 Materials

Ibuprofen (IBP) was used as the active pharmaceutical ingredient in this study. USP grade

IBP (Part no: 30-1192-1000GM) was purchased from Professional Compounding Chemists of

Australia Pty Ltd (PCCA, Matraville, NSW 2036), as a high purity racemate of (R)/(S)-(±)-[2-(4-

isobutyl-phenyl) propionic acid] with the empirical formula C13H18O2 and molecular weight

206.27. Pluronic F127 (Pl F127) is a surfactant copolymer (Poloxamer 407 NF, Part no: 302637-

500GM) and was purchased from PCCA (Matraville, NSW). Hydroxypropyl methyl cellulose/

hypromellose (HPMC), used as stabilizer was purchased from Sigma-Aldrich (09963-100 G,

Lot: BCBG6002V). D-mannitol (C6H8OH6), used as a cryoprotectant and bulking agent was

purchased from Sigma-Aldrich (Part No: M4-125-500 g, Lot no: SLBJ5312V). L-Leucine

(C6H13NO2) (Bioultra, ≥ 99.5%), an amphiphilic surfactant used as a dispersive adjuvant was

purchased from Sigma-Aldrich (Part No: 61819-100 G, Lot No: BCBM2322V).

Spectrophotometric grade ethanol was purchased from Sigma-Aldrich and deionised/Millipore

water is available in the laboratory. All the materials were used as received.

2.2 Methods

2.2.1 Preparation of HPMC solutions

Solutions containing 5% w/w HPMC were prepared by adding HPMC powder to a weight

of water required to make up to one tenth of the final weight. Vigorous agitation was used until

the powder was dissolved. The solution was then refrigerated for 24 hours at 4°C to allow

polymer hydration, made up to final weight and stored refrigerated for 72 hours prior to use 15

.

The final solvents of the required concentration of additives were prepared by taking the weighed

amount of Pl F127, leucine and mannitol powder, made up to the final weight with the 5%

HPMC solution and water/aqueous ethanol to give the additives in the concentration range


5

HPMC 0-0.8%, Pl F127 0-1.8%, leucine 0-1.5% and mannitol 0-9%. They were magnetically

stirred until complete dissolution was observed.

2.2.2 Anti-solvent precipitation crystallization (APC) method for particle preparation

The method for particle preparation was the anti-solvent precipitation crystallization (APC)

by a co-solvent technique 16

. This technique involves mixing of two different phases as

demonstrated in Fig. 1. The first phase (solvent phase) is ethanol with dissolved IBP (30-200

mg/g) depending on the batch size. The second phase (anti-solvent phase, water) in which IBP is

practically insoluble contains the dissolved additives. The crystallizer comprises an ultrasonic

bath (Soniclean 750 HT), the cooler (Julabo, FT 200), the constant temperature heating

immersion circulator ED (Julabo) and the overhead stirrer (Lab Co.).Various process parameters

(Table 1) including temperature, concentration of excipients (HPMC, Pl F127, leucine and

mannitol), batch size, use of ultrasound and stirring speed were varied to get the best size of the

micro/nanocrystals. The compositions of the prepared particles formulations are given in Table

2.

2.2.3 Freeze drying process for particle recovery

The micro/nano crystal product suspensions were centrifuged at 3500 rpm for 60 minutes

in Falcon tubes (10ml and 50ml) to remove non-adhering additives. The excess liquid was

discarded and the remainder was frozen using a deep freezer at -75ºC for 24 hours. These frozen

semisolids were freeze dried using a lyophilizer (Alpha 1-4 LD plus) at a temperature of -55°C

and vacuum 1.0 mbar absolute for 24 – 96 hours, depending on the sample volume.

2.2.4 Particle size reduction by mill micronizer

A McCrone Micronizing mill was used to mill raw IBP powder down to the similar size

obtained by controlled crystallization technique. Raw IBP powder was loaded (< 3 g) into the

micronizer vessels with zirconia beads. Water with > 300 ppm of detergent (2 mL) was used as a

fluid. Particles with the required size were found after 12 hours of milling. The samples were

then dried in an oven overnight at 40 °C.

2.3. Characterization of prepared particles

2.3.1. Density and flow property measurements


6

The density, angle of repose, Carr’s index and the Hausner ratio of the prepared IBP

crystals was determined by the methodology published elsewhere 17

. Using the Erweka tapped

density tester, the densities of powders were determined. After observing the initial powder

volume and mass, the graduated 5 mL measuring cylinder was mechanically tapped, and volume

readings were taken until no further volume change was observed. A 5 mL graduated cylinder

was filled with a mass of 1.3 ± 0.3 g of powder sample. Then the cylinder was secured in the

support of the tapping apparatus and 100, 500 and 1250 taps on the same powder sample were

carried out, and the corresponding volumes V100, V500 and V1250 were recorded to the nearest

graduated unit. In this work, the Carr’s index and Hausner ratio was calculated using measured

values of bulk density (��) and tapped density (��) as follows:

�� = 100 ×!"#$$%&'!()*+

!"#$$%& (1)

,�-�� .�� = !"#$$%&

!()*+ (2)

The flowability of the samples was determined using the generally accepted scale of flowability

for the Carr’s index and the Hausner ratio18

. Measurements were taken from three replicates of

each of the formulations.

2.3.2. Angle of repose

To form the angle of repose on a fixed base, a 5 mL beaker was used as a base (10 mm

diameter) to retain the powder (250 ± 0.5 mg). The powder was poured through a funnel (40 mm

diameter and 65 mm height). The funnel height was maintained at approximately 2 – 4 cm from

the top of the powder pile which is formed in order to minimise the impact of falling powder on

the tip of the cone. The angle of repose was determined by measuring the height of the cone of

powder and calculating the angle of repose, α, from the following equation:

tan234 =5675�

8.:×��; (3)

The degree of flowability was determined from the flow properties corresponding to the angles

of repose17

. Measurements were taken from three replicates of each of the formulations.

2.3.3. Particle size measurement


7

The particle size of the crystallized IBP was measured by the laser diffraction technique

using a Malvern Mastersizer 3000 equipped with a small volume dispersion unit. It is estimated

that the relative error in the volume median diameter (VMD) calculated by the Malvern

Mastersizer is ± 2% 19

. Particle size measurements for the finer nanocrystals were done using the

Zetasizer (NanoZS 90, Malvern Instruments, UK). The suspending media was the saturated IBP

solution prepared with an equivalent composition to the final crystallization media at

equilibrium. This media was used as the dispersant in the Malvern 3000 small volume (120 ml)

dispersion unit stirring at 2000 rpm. A small amount of prepared powder was dispersed in 5.0

mL of this dispersion media and sonicated for 5 minutes to ensure all particles dispersed in the

suspension. Few drops of this concentrated suspensions was added dropwise in the dispersion

unit until the obscuration reached the desired level. The refractive indexes used for IBP and the

dispersant were 1.43 and 1.33, respectively. The absorption index was 1.2. The mass median

diameter (MMD, D[v,0.5]) and volume mean diameter (D [4,3]) determined from the output of

the laser diffraction particle sizing were used as the major size parameter to characterize the

particle size distributions (PSD). Measurements were taken from three replicates of each of the

formulations.

2.3.4. Scanning electron microscope (SEM)

A Zeiss Sigma scanning electron microscope was used to investigate the morphological

properties (shape, size and surface) of the IBP crystals. The sample preparation involved fixing

the powder samples on to a metal stub with the aid of a double sided adhesive tape followed by

coating for 180 seconds with a LEICA EM SCD005 gold coater. Scanning electron microscopy

(SEM) was then carried out by loading the sample on the SEM working at 5 KV.

2.3.5. Drug loading determination

Quantification of the IBP content in the powder formulation was determined by UV

spectrophotometer (Thermo Scientific Evolution Array) at a wavelength of 264 nm. Samples

were prepared by dissolving a known 10-15 mg of powder formulation in a known 10 (± 1) g of

50% aqueous ethanol solvent system. Complete solution of IBP in the selected solvent system

was confirmed from the predetermined solubility results.

2.3.6 Differential scanning calorimetry (DSC)


8

Differential scanning calorimetry (DSC) experiments were carried out in a DSC Q100

from TA Instruments Explorer Q series. A known small amount of sample (<6 mg) was enclosed

in a hermetic sealed aluminum pan. A liquid nitrogen cooling system was used in order to reach

temperatures as low as - 42 °C. Processed and unprocessed IBP samples were scanned from 10

°C to 110 °C, using a heating rate of 10°C/min. All samples were analysed in triplicate. The

percent crystallinity is determined using equation 4:

% Crystallinity = ∆Hm / ∆Hm°× 100% (4)

The heats of melting, ∆Hm, determined by integrating the areas (J/g) under the peaks using TA

instrument Analysis 2000 software. The term ∆Hm° is a reference value and represents the heat

of melting of the 100% crystalline IBP. It was found that the melting enthalpy of the raw IBP

powder was 118.4 ± 7.3 J/g which is also in agreement with Nokhodchi and co-workers 3. This

value was used as the reference value to determine the percentage crystalline phase of IBP in the

processed formulations with different compositions of additive.

2.3.7. Powder X-ray diffraction (XRD)

The crystallinities of the unprocessed and processed IBP were evaluated using an X-ray

powder diffractometer (XRD). Samples were front pressed into low background quartz holders.

Diffraction patterns were collected in θ/2θ geometry on a PANalytical X’Pert Pro diffractometer

(Co Kα) using a W/Si parabolic mirror and 0.09° collimator before the point detector. A 0.25°

fixed divergence slit, 10 mm mask, 1.4 mm incident anti-scatter slit, and 0.04 rad pre and post

diffraction Soller slits were used. Patterns were collected from 3 – 75° 2θ at a step size of 0.02°

for 1 hr. The sample was spun during data collection. An instrument function was determined

from LaB6 (SRM 660a). Phase identification was performed with Highscore Plus (V4.5,

PANalytical) using the PDF4+ database (ICDD) and confirmed via Rietveld refinement with

TOPAS (V5, Bruker). Quantitative phase analysis was performed using TOPAS (v5, Bruker) via

the Rietveld method. An instrument function determined from LaB6 (NIST SRM 660a) was used

to model the profile shape. A Lorentzian crystallite size term was refined for each phase to

account for profile broadening. Refined terms included specimen displacement, scale factor for

each phase, and unit cell parameters for each phase. The amount of Pl F127 in some samples was

estimated by the degree of crystallinity method where the numerical area of each phase is used to


9

determine abundances. The Pl F127 was accounted for by modelling a peak at ca. 27° 2θ (most

obvious feature not modelled) and designating it as the amorphous phase. HPMC phase

abundance could not be quantified as its concentration in the formulation was too low to identify

in the XRD curves. XRD patterns of both the processed and unprocessed IBP were compared to

identify any alteration in their crystallinities.

2.3.8. Raman spectroscopy

A WITec Alpha 300 series Raman Microscope equipped with a 532 nm laser was used for

spectral analysis of the individual components and the powder formulations. The purpose of the

analysis was to identify the IBP component and additives and their distribution by recording

Raman maps over selected regions of the powder formulations. To prepare the sample for

mapping a stainless steel cup was piled slightly high with the formulation powder, which was

then lightly tamped down by placing a coverslip over the top of the cup. The purpose of the

coverslip was to establish a horizontal region in the sample that would remain in focus over the

region to be studied. Raman maps were measured by rastering in the horizontal plane in 0.5 µm

increments over a 20 x 20 µm region of the powder with the focal plane of the microscope set to

just beneath the coverslip. Spectra were recorded at each increment with the integration time of

1s and a laser power of 10 mW. The Raman microscope was calibrated to the 520.5 nm line of a

silicon wafer. The Zeiss 50× objective with a 0.7 NA used forms a confocal sample volume

approximating a cylinder of 0.5 µm diameter and 2-3 µm high. The mapping of the powder

components in the mixture and the spectral analysis including CLS calculations were performed

using WITec Control Four and Project Four software.

2.3.9. In vitro drug dissolution test

The USP paddle method (Pharma Test- DT 70) was adopted in 900 mL phosphate buffered

saline (PBS, pH 7.4) at 100 rpm and 37 °C for each sample. At the fixed time intervals of 2, 6,

10, 15, 20, 30, 60, 90 and 120 minutes, 5 mL aliquots of the release medium were withdrawn and

the same amount replaced with the fresh PBS. The drug content in the withdrawn aliquot was

analyzed by UV spectrophotometry at 221 nm. Percentage drug release versus time data were


10

plotted to establish drug release profiles from the formulations. Three replicates were undertaken

on each sample. A sample of milled pure IBP (12.5 mg; size 2.8 ± 0.1 µm) used as the control

and three processed formulations (F4, F6 and F10, Table 2) equivalent to 12.5 mg of pure IBP

were selected for dissolution studies as these processed formulations showed better flow

property.

2.3.10. Statistical analysis: All statistical analysis was performed using Microsoft Excel (2016).

3. Results and discussions

3.1 Formulations of the prepared particles

To evaluate the efficiency of the processed IBP powder in an APC process, 14 different

formulations were prepared based on the different batch size of crystallization, initial IBP

concentrations and additive concentrations. It is noted that the additives mentioned in Table 2 are

the percentages that were dissolved in the crystallization medium for the total batch size and are

not the additive contents of the crystal product. Each of the products from these formulations was

characterized for density, flowability, particle size distribution, morphology, and crystallinity.

F1, F2, F3, F6 and F7 formulations had different batch sizes from the usual 50g. IBP

concentration was varied from 0.3% to 2% in the formulations F1 to F6. Additive concentrations

(Pl F127, HPMC, L-leucine and D-mannitol) were varied in other runs. Initially, using different

batch size (10-50 g) the precipitation experiments were carried out at various stirring speeds

(500-2000 rpm) with different duration of mixing (30-60 minutes) to produce small particles.

Based on the initial runs (a large number of raw data are not presented in this article), the best

parameters were selected to produce reproducible particle size from a fixed batch of the

formulation containing the fixed amounts of different components. Finally, keeping the constant

solvent-anti-solvent ratio (1:9), the optimized process parameters demonstrated in Table 1 have

been selected from these initial studies for the rest of the experiments with varying

concentrations of other excipients as demonstrated in Table 2 to obtain similar particle size

presented in Table 3.


11

3.2 Particle size & size distribution

Particle size and size distribution of the prepared IBU crystals are presented in Table 3. The size

of particles was affected by the presence of different concentrations of surfactant Pl F127. The

decrease in size occurred with increased concentration of Pl F123 until 1.2% (F7) in the

formulations; however, the size increased at 1.8% of Pl F127 (Formulation 9; Suppl Fig. S1).

Both mannitol and PL F127 aid in reducing particle size. FPO and FMO are the formulations

without Pl F127 and D-mannitol respectively, and particles grew larger in these formulations due

to their absence. The literature has shown that mannitol prevents nanoparticle aggregation during

the drying process. The stabilization of particles during the freeze drying is proposed to occur by

sugars isolating individual particles in the unfrozen fraction, thereby preventing aggregation

during freezing above the glass transition temperature, Tg 20

. In this case, the transition of the

substance into a glass is not required for this effect and the spatial separation of particles within

the unfrozen fraction is sufficient to prevent aggregation20

. As a result, the particle size was

expected to remain stable with no further growth due to aggregation after the drying step.

3.3 Density, angle of repose & flowability

The estimation of the bulk density and tapped density (resulting in the Carr’s index and Hausner

ratio) and also the angle of repose for estimating flowability are given in Table 4. Flowability of

all the formulations was determined and the effect of the additive and drug concentration on it

was observed from the obtained results. The purpose of preparing all of these formulations was

to identify those formulations with smaller particle size for faster dissolution. Table 4 shows that

formulations F3, F4, F6, F10 and FMO had good flow properties (Angle of Repose, AR): 31-35;

Carr’s index (CI): 11-15; Hausner ratio (HR): 1.12-1.18), F1, F2 and FLO were passable and the

rest were poor (Table 4). Of those with good or passable flowabilities; the FPO and FMO

formulations had larger particle size (D[4,3] > 10 µm) compared to others. It was expected that

the additive concentration might have a significant effect on the flow properties of the prepared

IBP particles due to their hygroscopic nature. L-leucine is a well-accepted dispersive excipient

for use in pharmaceutical formulations and it has been found to improve the powder flowability

in several cases due to its anti-adhesive 21,22

functions and surfactant properties 2,23

. Hence, to see

the leucine effect on flowability, its concentration was increased gradually from 0%, through

0.9%, 1.2% to 1.5% in FLO, F7, F10 and F11 formulations, respectively. However, no specific


12

trend was observed in the flowability according to the values of Carr’s index, Hausner ratio and

angle of repose (as illustrated by the angle of repose, Table 4). Surprisingly, FLO has no leucine

but showed a better flowability than those of the formulations containing 1.5% leucine (F7 and

F11). An optimal amount of leucine is critical to achieving the desired properties of the leucine

coated particles, as excessive leucine decreases stability with no additional benefits 24

.

Interestingly, an increase in the Pl F127 concentration in the formulations FPO, F8, F7 and F9,

where concentration of PL F127 was increased from 0% to 0.6%, 1.2% and 1.8%, appeared to

influence the flowability negatively. A possible reason could be the decreased particle size with

increasing Pl F127 concentration, caused the flowability to decrease 25

. It has been reported that

the drug particle has a partial amorphous form due to the presence of Pl F127 26

, which might

have influenced the powder flow negatively. Thus, although leucine has remarkable effects,

other excipients used to prepare the IBP microparticles have no such effect on the flowability of

the prepared powders.

3.4 Particle Morphology by SEM

The particle morphology of the formulations was investigated by scanning electron

microscopy (SEM). The SEM images of raw IBP, milled raw IBP and all the formulations are

presented in Fig. 2. The crystal habit of IBP depends on the crystallization conditions such as the

solvent type and presence of additives2,27

. The SEM images show that the commercial raw IBP is

needle-shaped, whereas the particles crystallized in the presence of various additives are mostly

of irregular shapes with pores in the agglomerates. In the case of formulation F5, the

crystallization was carried out in the absence of leucine and mannitol, which resulted in a

different morphology, with the crystals having rough surfaces comprising flat-shaped IBP

particles sticking together to make bigger particles. As the SEM images represent the dried form

of formulation, it is very likely the particles in F5 formulation aggregated/ agglomerated into

large irregular shapes due to drying without the leucine and mannitol. The morphology of the

formulations F6, F7, F8, F9, F10, F11, FPO, FLO and FMO with low IBP concentration (0.3%)

seems to be the agglomerates of IBP microparticles adhering to each other. The IBP particles in

FMO are seen clearly in the tight agglomerated form of chunky shaped crystals. The images

revealed that the needle shaped crystals actually represents the crystal habit of D-mannitol in the


13

powder mixtures. However, the IBP particle morphology was significantly influenced by both

the additive composition and concentration.

3.5 Raman mapping for excipients distribution in the microcrystals of IBP

The particle morphology was investigated using Raman spectroscopy to identify each component

individually and the distribution of the drug particle with the additives in the powder mixture is

represented in Fig. 3. Image A is formed by merging the component images to give an overall

impression of distribution of all components in the co-crystal. The individual components such

as red representing leucine (B); magenta-pluronic F127 (C), green-HPMC (D), yellow-IBP (E),

and aqua-mannitol (F) are also shown. The Raman images revealed that IBP drug particles are

surrounded by the additives used. The distribution of Pl F127, HPMC and L-leucine around the

IBP particle, reflected the interactions of those excipients with the active drug during the APC

process.

To explore the possible associations between the excipients and IBP, correlation diagrams were

prepared by plotting the relative concentration of each component vs the IBP concentration for

each mapped point. Results are only shown for those points where significant IBP was present.

Correlation plots for the representative formulation F6 are given in Figs. 4 (A-D). A broad trend

to higher leucine corresponding to higher IBP concentrations seen in Figs 4A suggesting a

positive correlation between the relatively hydrophobic components leucine and IBP. The Pl

F127 is closely associated with IBP without significant correlation (4B). The presence of

hydrophilic HPMC (4C) is poorly distributed in regions of high IBP, suggested a negative

correlation between the cellulose components with extremely hydrophobic IBP. However, the

hydrophilic mannitol (4D) is distributed in the region of high IBP and very closely associated

with the drug particle presumably due to hydrogen bonding. These outcomes helped get a clear

understanding on the solubility/dissolution behavior of the prepared IBP microcrystals.

3.6 Crystallinity

The crystallinity of the raw IBP powder, milled IBP powder, additives and the powder

formulations was examined using differential scanning calorimetry (DSC). The DSC data are

presented in Figs. 5 A & B). All the formulations produced an endothermic peak in the range of

74–78°C, which are in agreement (within ± 2.0 °C) with the previous reports which suggest that


14

IBP exists as crystalline solid exhibiting a typical melting range of 75–77 °C; however, the

melting point of pure IBP is 77.5ºC28

. The melting point for pure IBP, HPMC, Pl F127, mannitol

and leucine had shown a sharp peak before the melting point of the pure IBP drug (Fig. 5A). The

additive melting peaks confirmed that the presence of mannitol and leucine would not interfere

with the pure IBP melting peak identification in DSC, but HPMC and Pl F127 could. Fig. 5B

represents the DSC curves for raw and milled IBP and all the prepared formulations where the

endothermic peaks for all the formulations, within the melting point range for pure crystalline

IBP; however, the height of the peaks changed with respect to the IBP content in the

formulations. The pure, milled IBP particle and formulations with high concentrations of IBP

(F4 and F5) showed very sharp peaks, whereas the crystals with other additives and low

concentrations of IBP showed smaller and broader peaks suggesting that the IBP in the prepared

formulations are both in crystalline and amorphous forms. The pure IBP showed 100%

crystallinity, milled IPB produced 94.7% crystallinity due to crushing; however, formulations

(F4 and F5) with 2.0% IBP at varying concentrations of other excipients showed 96-99%

crystallinity (Suppl Table S1). Other formulations with low concentrations of IBP in presence of

other excipients at varying concentrations produced more or less 50% amorphous particles.

3.7 Dissolution studies

The dissolution profile of the milled raw IBP powder was much slower compared to those

of the prepared formulations (Fig. 6). Theoretically, it was expected that the smallest particle size

of the milled ibuprofen (2.8µm, Table 3) compared to the prepared particles would have a higher

surface area which could enhance the solubility. However, this did not happen presumably due to

the strong hydrophobic nature of the drug particle. The prepared IBP micropartilces showed

faster drug release/dissolution (Fig 6). It can be noted that the drug release from F4, F6 and F10

were 56, 64 and 96%, respectively in first two minutes. The results indicated that the dissolution

rate of the prepared formulations was significantly faster than that of the milled pure IBP (<50%

in 2 minutes). The formulations with the higher initial IBP and lower additive concentrations

have shown a comparatively lower dissolution rate in the first two minutes. Formulation F10

showed the highest drug release (96%) in the first two minutes. The possible reason could be the

presence of a higher content of additives (Pl F127 1.2%, L-leucine 1.2%, D-mannitol 9.0% and

HPMC 0.2%, Table 2) and very low initial drug load (0.3%) in the formulation F10 which


15

favoured the dissolution process by decreasing drug-drug cohesion and increasing the drug-

solvent interaction. The Raman images (Fig. 3) clearly demonstrated that a large number of

excipients especially the amphiphilic leucine and hydrophilic HPMC and D-mannitol containing

a large number of hydrophilic hydroxyl groups are distributed around the IBP particle in the

microcrystals. This suggests that the hydroxyl groups of these excipients enhanced the

dissolution of the drug by increasing the drug-solvent interaction. The hydrophilic HPMC (Fig.

4C) is poorly associated in regions of extremely hydrophobic IBP. However, because of the

amorphous character and extensive hydrogen-bonded network, it may help increase the

dissolution by interacting with solvent through hydrogen bonding. In contrast, the hydrophilic

mannitol (Fig. 4D) is closely associated with IBP which presumably led to the enhanced

dissolution by hydrogen bonding.The enhanced dissolution of the prepared formulations

especially the formulation F4 indicated that the presence of the hydrophilic surfactant pluronic

(Fig. 4B) around the IBU particles (Fig. 3 E) strongly promoted drug wettability by reducing the

surface tension of the hydrophobic IBP particles. Additionally, the presence of amphiphilic

surfactant L-leucine in the complex crystal played a significant role in increasing dissolution of

the drug possibly due to the orientation of hydroxyl groups towards the aqueous solvent, which

increased the particle-solvent interactions and improved the dissolution process29

. The presence

of hydrophilic surfactant pluronic and amphiphilic leucine around the IBP crystal strongly

promoted the wettability by reducing the surface tension of the hydrophobic IBP particles

leading to increased dissolution. Additionally, the amorphous IBP particles in the prepared

formulations especially the sample F6 (52% crystalline, Suppl Table S1) containing the low

concentration of IBP (1.0%) showed higher dissolution (Fig. 6), as this particle in the amorphous

state contained limited crystal lattice interactions or bonds that needed to break for the drug to

enter solution leading to higher dissolution compared to that of the pure crystals, which has

greater intermolecular forces in the crystal. Furthermore, the formation of pores in the complex

microparticles (SEM images Figs 2) increased the surface area and wettability of drug particles

and led to increasing the solubility/dissolution. On the other hand, the relatively higher cohesive

forces among the milled pure IBP particles with the smallest size (2.8µm) potentially influenced

the overall dissolution negatively due to the extreme hydrophobic nature30

. The maximum

release from milled pure IBP was 71%, whereas, from the prepared formulations F4, F6 and F10


16

were 100, 87% and 96%, respectively in 20 minutes. This promising outcome of dissolution

studies of the prepared microcrystals ensured the superiority of the prepared IBP over pure

milled drug particles. Therefore, it can be emphasized that the prepared formulations are better

than the milled IBP in terms of the dissolution behaviour. The faster dissolution performance of

the prepared powder formulations would be useful for the development of dosage forms like

tablet, capsule, suspensions, and formulation for inhalation to achieve an improved

bioavailability.

4. Conclusion

This study reports the preparation of the hydrophobic IBP microparticles along with other

additives (HPMC, mannitol, leucine and pluronic) by a controlled crystallization technique with

enhanced dissolution profile. The prepared IBP microparticles showed higher dissolution

compared to that of the smaller sized original milled IBP particles as the presence of hydrophilic

excipients in the formulation played a vital role. The Raman spectroscopy used for the first time

in this study to investigate the interactions and distribution of the IBP with other additives

present in the microcrystals, clearly demonstrated that a large number of excipients especially the

amphiphilic leucine and hydrophilic HPMC and D-mannitol containing a large number of

hydrophilic hydroxyl groups are distributed around the IBP particle. The hydroxyl groups of

these excipients enhanced the dissolution of the drug by increasing the drug-solvent interaction.

The hydrophilic HPMC and mannitol associated in regions of extremely hydrophobic IBP in the

in the microcrystals, they presumably increase the dissolution process by interacting with solvent

through hydrogen bonding. The Raman mapping technique, which provides the distribution of

different components in the microcrystals, gave an insight into the enhanced

solubility/dissolution behaviour of the prepared IBP microparticles. Such information will be

useful for developing pharmaceutical formulations with better solubility and dissolution

properties of hydrophobic drugs.

Acknowledgement: The authors gratefully acknowledge the financial support of PhD

scholarship for Afrina Afrose from Queensland University of Technology (QUT, Australia. The

Raman data reported was obtained using the resources of the Central Analytical Research


17

Facility (CARF) at the Institute for Future Environments. Access to CARF is supported by

generous funding from the Science and Engineering Faculty (QUT).

The authors declare no conflict of interest.


18

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Tables

Table 1: Anti-solvent precipitation crystallization (APC) process parameters and optimized

range of conditions for preparing respirable IBP particles.

Process parameters Condition

Batch size 10 – 50 g

Solvent-anti-solvent ratio (solv/anti-solv) 1 : 9 (fixed)

Stirring speed, rpm 600 - 1200

Temperature 25 - 30°C

Ultrasound application 50Hz, max. pulse swept power 180W

Duration of mixing 30 - 60 minutes

Drug concentration in organic phase 0.3-2%

Pl F127 concentration 0-1.8%

HPMC concentration 0-0.8%

Leucine concentration 0-1.5%

Mannitol concentration 0-9%


Table 2. Composition of the different formulations and the amount of additives.

Formulation

name

Batch

size, g

IBP*

conc.%

Leucine*,%,

(w/w)

Mannitol*,%,

(w/w)

HPMC*,%,

(w/w)

Pl

F127*,%,

(w/w)

Drug

loading

(%)

F1 10 1 1.3 4.5 0.4 1.8 72.7

F2 30 1 0.9 4.5 0.7 1.3 77.9

F3 10 1 0.9 4.5 0.7 1.3 90.3

F4 50 2 1 8.4 0.1 0.9 99.8

F5 50 2 0 0 0.1 0.9 100.0

F6 10 1 0.9 4.5 0.6 1.2 83.3

FPO 50 0.3 0.9 9.0 0.2 0 84.7

F7 10 0.3 0.9 9.0 0.2 1.2 53.2

F8 50 0.3 0.9 9.0 0.2 0.6 74.6

F9 50 0.3 0.9 9.0 0.2 1.8 43.9

FLO 50 0.3 0 9.0 0.2 1.2 83.9

F10 50 0.3 1.2 9.0 0.2 1.2 73.5

F11 50 0.3 1.5 9.0 0.2 1.2 52.7

FMO 50 0.3 0.9 0 0.2 1.2 99.9

*These are the percentages of drug/additives in total amount of crystallization batch size. F1 to F11 are

the formulations with different components. FPO represents the formulation without pluronic, FLO represents

the formulation without leucine, and FMO indicates the formulation without mannitol.


Table 3: Particle sizes and distributions of various IBP formulations (units µm± SD, n=3).

Formulation D[v,0.1] D[v,0.5] D[v,0.9] D[4,3]

F1 2.9 ± 0.1 6.9 ± 0.1 20.4 ± 2.9 9.6 ± 0.8

F2 3.5 ± 0.1 8.5 ± 0.1 18.0 ± 0.2 9.7 ± 0.1

F3 4.6 ± 0.1 17.1±0.5 47.9 ± 1.4 22.0 ± 0.4

F4 3.3 ± 0.6 6.2 ± 0.2 9.3 ± 0.3 6.3 ± 0.2

F5 4.2 ± 0.3 7.2 ± 0.5 11.3 ± 1.5 7.5 ± 0.5

F6 2.9 ± 0.5 5.9 ± 0.8 10.7 ± 1.6 6.5 ± 1.1

FPO 6.1 ± 0.1 11 ± 0.6 18.7 ± 1.3 12.34 ± 1.2

F7 1.1 ± 0.1 3.9 ± 0.4 9.0 ± 0.9 5.1 ± 0.9

F8 3.7 ± 0.1 6.8 ± 0.1 11.9 ± 0.1 7.4 ± 0.1

F9 1.3 ± 0.1 6.1 ± 0.1 12.8 ± 0.3 6.7 ± 0.1

FLO 3.2 ± 0.1 6.1 ± 0.1 10.1 ± 0.3 6.6 ± 0.6

F10 1.4 ± 0.2 6.4 ± 0.3 12.9 ± 0.5 7.1 ± 0.2

F11 1.2 ± 0.0 5.4 ± 0.6 11.8 ± 0.7 6.4 ± 0.4

FMO 3.7 ± 0.1 8.7 ± 0.1 28.2 ± 1.2 20.9 ± 1.3

Milled IBP 1.2 ± 0.1 2.8 ± 0.1 4.45 ± 0.2 2.8 ± 0.1


Table 4: Powder flow properties obtained from different formulations (Mean ± SD, n=3)

Formulation Bulk density

(g/ml)

Tapped

density (g/ml)

Carr’s

index (%)

Hausner

ratio

Angle of

Repose (°)

Flowability

F1 0.28 ± 0.02 0.36 ± 0.03 21.5 ± 0.3 1.27 ± 0.01 44.0 ± 0.1 Passable

F2 0.24 ± 0.03 0.28 ± 0.01 23.7 ± 1.0 1.31 ± 0.02 42.0 ± 1.7 Passable

F3 0.27 ± 0.04 0.35 ± 0.01 11.1 ± 1.0 1.12 ± 0.01 34.2 ± 1.8 Good

F4 0.29 ± 0.01 0.34 ± 0.01 13.2 ± 0.8 1.15 ± 0.01 33.9 ± 1.1 Good

F5 0.27 ± 0.03 0.35 ± 0.01 27.1 ± 0.8 1.37 ± 0.01 50.4 ± 0.2 Poor

F6 0.26 ± 0.01 0.30 ± 0.02 13.9 ± 0.7 1.16 ± 0.01 36.5 ± 1.3 Good

FPO 0.20 ± 0.02 0.29 ± 0.01 30.8 ± 0.7 1.44 ± 0.01 53.3 ± 0.4 Poor

F7 0.18 ± 0.05 0.26 ± 0.02 30.1 ± 0.6 1.43 ± 0.01 53.7 ± 1.1 Poor

F8 0.22 ± 0.03 0.34 ± 0.04 31.1 ± 0.4 1.45 ± 0.01 53.0 ± 0.7 Poor

F9 0.21 ± 0.02 0.32 ± 0.01 37.5 ± 0.7 1.60 ± 0.02 60.3 ± 1.4 Very poor

FLO 0.29 ± 0.001 0.36 ± 0.01 18.3 ± 0.6 1.22 ± 0.01 37.5 ± 0.4 Fair

F10 0.14 ± 0.01 0.16 ± 0.02 13.1 ± 1.0 1.15 ± 0.01 33.9 ± 2.0 Good

F11 0.18 ± 0.05 0.21 ± 0.03 26.9 ± 0.5 1.37 ± 0.01 53.3 ± 1.1 Poor

FMO 0.11± 0.01 0.13 ± 0.02 13.7 ± 0.5 1.16 ± 0.01 34.7 ± 1.9 Good

Milled IBP 0.13 ± 0.03 0.17 ± 0.05 22.7 ±1.0 1.29 ± 0.02 41.8 ± 0.5 Passable


Legend for Figures

Fig. 1. Anti-solvent precipitation crystallization (APC) process for the preparation of IBP

microparticles.

Fig.2. Particle morphology of the raw and milled IBP and formulations F1, F2, F3, F4, F5,

F6, F7, F8, F9, F10, F11, FLO, FMO and FPO in scanning electron microscopy

(Magnification: 5.00 K X).

Fig.3. Raman images of the microcrystal (A) of a representative formulation F6 with the

individual components separately coloured: Red for leucine (B); magenta for pluronic F127

(C), green for HPMC (D), yellow for IBP (E), and aqua for mannitol (F). These images

demonstrate the way that the drug crystal is surrounded by the excipients.

Fig. 4. Raman correlation plots of formulation F6. A) leucine vs IBP; B) Pl F127 vs IBP; C)

HPMC vs IBP; and D) mannitol vs IBP.

Fig. 5. DSC curves for (A) Pluronic F127, HPMC, L-leucine, D-mannitol, raw IBP; (B)

milled IBP, F1, F2, F3, F4, F5. F6, F7, F8, F9, F10, F11, FPO, FLO, FMO and raw IBP.

Fig. 6. In vitro dissolution of milled raw IBP powder and some formulations prepared by

APC process.

Supplementary Figure

Fig. S1. The effect of Pl F127 concentration on particle size. The crystallization solution

contains 0.3% IBP;0.2% HPMC; 0.9% leucine; 9.0% mannitol; 50 g batch (except F7 which

was 10 g).


210x297mm (200 x 200 DPI)


210x297mm (200 x 200 DPI)


210x297mm (200 x 200 DPI)


210x297mm (200 x 200 DPI)


210x297mm (200 x 200 DPI)


210x297mm (200 x 200 DPI)


210x297mm (200 x 200 DPI)


210x297mm (200 x 200 DPI)


Supplementary Table

Table S1: DSC data obtained for various formulations [mean ± SD, n=3]

Formulation Peak (°C) Enthalpy (jg-1) *Crystalline IBP (%)

*Pure IBP 77.1 ± 0.5 118.4 ± 7.3 100

Milled IBP 74.2 ± 0.7 111.8 ± 0.9 94.7 ± 0.9

F1 74.6 ± 0.3 40.02 ± 6.8 33.8 ± 5.7

F2 76 ± 1.4 75.4 ± 6.5 63.7 ± 5.5

F3 77.1 ± 0.1 84.8 ± 3.3 71.6 ± 2.8

F4 76.3 ± 0.4 117.4 ± 2.0 99.1 ± 1.7

F5 76.5 ± 0.8 113.3 ± 4.7 95.7 ± 4.0

F6 76.0 ± 0.5 61.2 ± 5.2 51.7 ± 4.4

FPO 77.1 ± 0.9 63.7 ± 4.6 53.7 ± 3.9

F7 75.0 ± 0.4 30.6 ± 2.8 25.8 ± 2.4

F8 76.3 ± 1.3 75.6 ± 4.1 63.7 ± 3.4

F9 74.6 ± 0.3 13.0 ± 1.1 11.0 ± 1.0

FLO 75.4 ± 1.2 65.1 ± 2.4 55.0 ± 2.1

F10 75.6 ± 0.4 47.4 ± 1.5 40.0 ± 1.3

F11 75.3 ± 0.3 34.5 ±1.4 29.1 ± 1.2

FMO 78.0 ± 3.8 83.2 ± 7.4 70.3 ± 6.2

HPMC 55.5 ± 0.8 243.8 ± 1.2 0

Pl F127 77.5 ± 0.4 134.1 ± 2.3 0

Mannitol 165.9 ± 0.5 303.3 ± 1.4 0

Leucine >300 Not determined 0


Documents

Afrose, Afrina, White, Edward, Howes, Tony,George, Graeme ...€¦ · 5 HPMC 0-0.8%, Pl F127 0-1.8%, leucine 0-1.5% and mannitol 0-9%. They were magnetically stirred until complete